International Journal of Organic Chemistry, 2013, 3, 65-72
Published Online November 2013 (http://www.scirp.org/journal/ijoc)
http://dx.doi.org/10.4236/ijoc.2013.33A007
Open Access IJOC
Synthesis, Characterization, Anti-Bacterial and
Anti-Fungal Activities of New Quinoxaline
1,4-di-N-Oxide Derivatives
Dalia Hussein Soliman
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
Email: odihss@yahoo.com
Received September 4, 2013; revised October 8, 2013; accepted October 24, 2013
Copyright © 2013 Dalia Hussein Soliman. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
A new series of quinoxaline 1,4-di-N-oxides were synthesized and evaluated for their antibacterial and antifungal ac-
tivities. The best result was demonstrated by 3-amino-N-(4-methoxyphenyl)-2-quinoxalinecarboxamide 1,4-di-N-oxide
4e, MIC (0.24 µg/ml) against Aspergillus fumigatus, and (0.12 µg/ml) against Streptococcus pneumonia.
Keywords: Quinoxaline 1,4-di-N-Oxide; Beirut Reaction; Benzofuroxanes; β-Ketoamide;
β-Cyanoamide; Antibacterial; Antifungal
1. Introduction
There is a growing need to develop new antibacterial
agents in order to overcome the emergence of bacterial
resistance to antibiotic therapy. Quinoxaline 1,4-di-N-
oxide is a nucleus that displays a wide range of activities.
Quinoxaline 1,4-di-N-oxides and their derivatives demon-
strated excellent activities as antibacterial [1-5], antiplas-
modial [2,6,7], antifungal [8] and as antimycobacterial
[9-13]. Encouraged by these reported activities and with the
aim of searching for new, broad spectrum and more potent
antimicrobial compounds which can improve the current
chemotherapeutic treatments, 20 new quinoxaline 1,4-di-N-
oxide derivatives were synthesized and evaluated. In most
of the quinoxaline 1,4-di-N-oxides reported as antibacte-
rial and/or antimycobacterial, the carboxamide in position 2
was a common feature [11,13]. Therefore, the newly syn-
thesized compounds 3a-n possessed a carboxamide in
position 2 that was substituted with various aryl moieties
bearing both electron-withrawing and electron donating
groups. This series of quinoxaline carboxamide was pre-
pared from unsubstituted benzofuroxane, 3a-g to be com-
pared to their 7-chloro counterparts 3h-n in order to study
the effect of substitution of chlorine atom in position 7.
2. Results and Discussion
2.1. Chemistry
The synthetic route for the preparation of the quinoxaline
1,4-di-N-oxide were obtained by the Beirut reaction[14,
15]. This reaction, which has been referred to as the Bei-
rut reaction, is an excellent method for preparing these
heterocyclic compounds. Benzofuroxanes 1a,b were ob-
tained by previously described methods [13,16]. Thus,
the appropriate BFO reacted with the corresponding
β-ketoamide (2a,b) or β-cyanoamide (3a-n) in the pres-
ence anhydrous potassium carbonate as a catalyst
(Scheme 1). The use of inorganic catalyst for this reac-
tion provided better means of purification with consid-
erably good yields, whereas the reaction of BFO with
cyanoacetamide was better carried out using triethyl-
amine as a catalyst and dimethylfomamide as the solvent.
These two intermediates, β-ketoamide and β-cyanoamide,
were prepared by condensation of either ethylacetoace-
tate or ethylcyanoacetae with different amines [11,12,17].
When the target compounds were prepared from mono-
substituted-BFO, the formation of isomeric quinoxaline
1,4-di-N-oxides was observed. In most cases, the 7-sub-
stituted isomer prevailed over 6-substituted isomer, and
when the methoxy substituted quinoxalines were pre-
pared, only the 7-isomer was obtained, as previously de-
scribed [18,19].
Moreover, in order to investigate the antimicrobial ac-
tivity of fused tricyclic and tetracyclic di-N-oxides de-
rived from quinoxaline 1,4-di-N-oxide the phenazine
derivative 5 and the indenoquinoxaline 6 were synthe-
sized. The Beirut reaction was similarly followed using
D. H. SOLIMAN
66
Scheme 1. Synthe sis of target compounds. Conditions: (i): EtOH, K2CO3; (ii): CNCH2CONH2, DMF/TEA 0˚C; (iii) dimidone,
ammonia gas, EtOH, 40˚C, 12 hr; (iv) indanedione, morpholine, EtOH, stirring, 12 hr.
cyclic diketones such as indandione or dimidone. All the
prepared compounds were characterized in light of their
microanalysis and spectral data including IR, 1H NMR
and mass spectrum.
2.2. Microbiology
The newly synthesized compounds were tested for their
antibacterial and antifungal activities. These assays were
performed at the Regional Center for Mycology and Bio-
technology, Antimicrobial unit test organisms, Cairo,
Egypt. Compounds 4i and 4k were previously synthe-
sized [17] but they were prepared again in order to per-
form the microbiological testing on them and compare
them to their unsubstituted counterparts. The tested com-
pounds were evaluated for their antifungal activity
against (Aspergillus fumigatus (RCMB 02568), Synce-
phalastrum racemosum (RCMB 05922), Geotricum
candidum (RCMB 05097) Candida albicans (RCMB
05036)). The antibacterial activity was evaluated against
(Gram positive: Streptococcus pneumonia (RCMB
010010), Bacillis subtilis (RCMB 010067)) and Gram
negative (Pseudomonas aeruginosa (RCMB 010046),
Escherichia coli (RCMB 010052)). Amphotericin B was
taken as a reference for the antifungal effect, while am-
picillin was the standard used for the evaluation of anti-
bacterial activity against gram positive bacteria and gen-
tamicin was used as a standard in assessing the activity
of the tested compounds against gram negative bacteria.
The results expressed as the mean zone of inhibition in
mm ± standard deviation beyond well diameter (6 mm)
produced on the microorganisms using (10 mg/ml) con-
centration of tested samples, shown in Table 1.
The initial screening of the tested compounds showed
promising activity of some of the compounds which en-
couraged the determination of their minimum inhibitory
concentration (MIC) (Table 2). The best results were
demonstrated by compound 4e both as antifungal and
against gram positive bacteria, it possessed double the
activity of the standard, amphotericin B against Asper-
gillus fumigatus, (0.24 and 0.49 µg/ml, respectively)and
ampicillin against Streptococcus pneumonia (0.12 and
0.24 µg/ml, respectively). This compound has also
displayed 4 times the activity of amphotericin B against
Syncephalastrum racemosum, (7.81, 1.95 µg/ml, respec-
tively). Other derivatives also possessed remarkable ac-
tivity against Syncephalastrum racemosum as 4i which
displayed double the inhibitory effect of the standard (3.9,
7.81 µg/ml, respectively), while 2a and 6 were as active
as the amphotericin B. Moderate activity against gram
positive bacteria Streptococcus pneumonia was also
demonstrated by compounds 3b and 6. However, this
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D. H. SOLIMAN 67
Table 1. Antifungal and antibacterial activity of the new ly synthe sized compounds.
Sample
Tested microorganisms
2a 3a 3b 4a 4b 4c St.
FUNGI Amphotericin B
Aspergillus fumigatus
(RCMB 02568) 21.6 ± 0.36a 17.6 ± 0.5821.3 ± 0.4417.6 ± 0.5818.9 ± 0.4418.2 ± 0.25 23.7 ± 0.1
Syncephalastrum racemosum
(RCMB 05922) 19.2 ± 0.44 15.6 ± 0.4420.1 ± 0.5816.3 ± 0.2517.2 ± 0.6316.9 ± 0.34 19.7 ± 0.2
Geotricum candidum
(RCMB 05097) 22.4 ± 0.58 19.2 ± 0.3723.2 ± 0.2520.3 ± 0.3820.3 ± 0.2516.5 ± 0.58 28.7 ± 0.2
Candida albicans (RCMB 05036) NAb NA NA NA NA NA 25.4 ± 0.1
Gram Positive Bacteria: Ampicillin
Streptococcus pneumonia
(RCMB 010010) 21.6 ± 0.63 20.3 ± 0.4423.3 ± 0.6320.3 ± 0.4318.3 ± 0.6316.0 ± 0.44 23.8 ± 0.2
Bacillis subtilis (RCMB 010067) 22.8 ± 0.32 22.4 ± 0.2524.1 ± 0.4421.4 ± 0.5320.4 ± 0.4418.3 ± 0.67 32.4 ± 0.3
Gram negativeBacteria: Gentamicin
Pseudomonas aeruginosa
(RCMB 010046) 18.2 ± 0.58 10.2 ± 0.5818.3 ± 0.6315.2 ± 0.5810.6 ± 0.62NA 22.6 ± 0.1
Escherichia coli (RCMB 010052) 20.8 ± 0.46 18.6 ± 0.4421.3 ± 0.3718.3 ± 0.2513.8 ± 0.5813.0 ± 0.46 23.2 ± 0.3
Sample
Tested microorganisms
4d 4e 4f 4h 4i St.
FUNGI Amphotericin B
Aspergillus fumigatus
(RCMB 02568) 14.2 ± 0.33 24.2 ± 0.44 12.6 ± 0.25 18.9± 0.44 22.3 ± 0.34 23.7 ± 0.1
Syncephalastrum racemosum
(RCMB 05922) 13.1 ± 0.25 20.9 ± 0.58 10.3 ± 0.58 17.2 ± 0.36 20.3 ± 0.25 19.7 ± 0.2
Geotricum candidum
(RCMB 05097) 19.8 ± 0.34 25.3 ± 0.37 15.2 ± 0.38 20.4 ± 0.58 23.4 ± 0.58 28.7 ± 0.2
Candida albicans (RCMB 05036) NA NA NA NA NA 25.4 ± 0.1
Gram Positive Bacteria: Ampicillin
Streptococcus pneumonia
(RCMB 010010) 16.2 ± 0.15 24.2 ± 0.44 14.6 ± 0.43 21.2 ± 0.63 21.3 ± 0.44 23.8 ± 0.2
Bacillis subtilis (RCMB 010067) 19.8 ± 0.42 26.4 ± 0.58 16.2 ± 0.53 22.4 ± 0.32 23.4 ± 0.67 32.4 ± 0.3
Gram negative Bacteria: Gentamicin
Pseudomonas aeruginosa
(RCMB 010046) NA 20.3 ± 0.44 NA NA 12.3 ± 0.58 22.6 ± 0.1
Escherichia coli (RCMB 010052) 17.4 ± 0.53 21.3 ± 0.63 11.2 ± 0.25 20.6 ± 0.46 20.6 ± 0.46 23.2 ± 0.3
Sample
Tested microorganisms
4j 4l 4m 4n 6 St.
FUNGI Amphotericin B
Aspergillus fumigatus
(RCMB 02568) 17.6 ± 0.58 20.6 ± 0.25 NA 15.9 ± 0.25 20.6 ± 0.33 23.7 ± 0.1
Syncephalastrum racemosum
(RCMB 05922) 19.4 ± 0.25 18.3 ± 0.34 NA 16.1 ± 0.44 19.2 ± 0.25 19.7 ± 0.2
Open Access IJOC
D. H. SOLIMAN
68
Continued
Geotricum candidum
(RCMB 05097) 19.9 ± 0.38 21.6 ± 0.58 NA 19.2 ± 0.63 22.4 ± 0.34 28.7 ± 0.2
Candida albicans (RCMB 05036) NA NA NA NA NA 25.4 ± 0.1
Gram Positive Bacteria: Ampicillin
Streptococcus pneumonia
(RCMB 010010) 17.7 ± 0.43 21.2 ± 0.44 12.3 ± 0.44 19.6 ± 0.25 22.4 ± 0.15 23.8 ± 0.2
Bacillis subtilis (RCMB 010067) 19.3 ± 0.53 22.6 ± 0.67 14.6 ± 0.63 20.3 ± 0.63 23.2 ± 0.42 32.4 ± 0.3
Gram negativeBacteria: Gentamicin
Pseudomonas aeruginosa
(RCMB 010046) NA NA NA NA 16.8 ± 0.37 22.6 ± 0.1
Escherichia coli (RCMB 010052) 13.7 ± 0.25 19.9 ± 0.46 14.4 ± 0.25 16.9 ± 0.44 20.4 ± 0.53 23.2 ± 0.3
aData are expressed in the form of mean zone of inhibition in mm ± standard deviation beyond well diameter (6 mm) produced on a range of environmental and
clinically pathogenic microorganisms using (10 mg/ml) concentration of tested samples; bNA: No activity.
Table 2. Antimicrobial and antifungal activity as MICS (µg/ml) of tested compounds against tested microorganisms.
MIC (µg/ml)a
Sample
Tested microorganisms 2a 3a 3b 4a 4b 4e 4h 4i 4l 6
St.
FUNGI Amphotericin B
Aspergillus fumigatus
(RCMB 02568) 0.98 31.25 1.9531.2531.250.247.810.981.95 3.9 0.49
Syncephalastrum racemosum
(RCMB 05922) 7.81 62.5 3.9 62.562.51.9531.253.9 15.63 7.81 7.81
Geotricum candidum
(RCMB 05097) 0.98 7.81 0.493.9 3.9 0.123.9 0.491.95 0.98 0.03
Candida albicans (RCMB 05036) NA NA NA NA NA NA NA NA NA NA 0.12
Gram Positive Bacteria: Ampicillin
Streptococcus pneumonia
(RCMB 010010) 1.95 3.9 0.493.9 15.630.121.951.951.95 0.98 0.24
Bacillis subtilis (RCMB 010067) 0.98 0.98 0.241.953.9 0.060.980.490.98 0.49 0.007
Gram negativeBacteria: Gentamicin
Pseudomonas aeruginosa
(RCMB 010046) 15.63 500 15.63125 500 3.9 NA 125 NA 31.25 0.98
Escherichia coli (RCMB 010052) 3.9 15.63 1.9515.63125 1.953.9 3.9 7.81 3.9 0.98
aMinimum inhibitory concentration (µg/ml); bNA: No activity.
series of compounds had no effect on Candida albicans
and were also ineffective against gram negative bacteria.
Moreover, compounds 2b, 4g, 4k and 5 showed no ac-
tivity against most of the tested strains.
A simple SAR study reveals certain features that could
improve the activity such as the amino group in position
3, quinoxaline unsubstituted in position 6, the electron-
donating substituents on the phenyl amino displayed bet-
ter effect than electron withdrawing. As for the fused
derivatives the tetracyclic indenoquinoxaline 6 was better
than the tricyclic phenazine 5.
3. Conclusion
In this study, certain novel quinoxaline 1,4-di-N-oxide
derivatives were synthesized and evaluated for their ant-
bacterial and antifungal activities. The 3-amino-N-(4-
methoxyphenyl)-2-quinoxalinecarboxamide 1,4-di-N-
oxide 4e demonstrated the best result with MIC 0.12
µg/ml compared to ampicillin (0.24 µg/ml) against Strep-
tococcus pneumonia and MIC 0.24 µg/ml compared to
Amphotericin B (0.49 µg/ml) against Aspergillus fumi-
gatus. However, there series of compounds were inactive
Open Access IJOC
D. H. SOLIMAN 69
against gram negative bacteria as well as candida albi-
cans
4. Experimental
4.1. Chemistry
4.1.1. Chemical Met hods
All the solvents used were commercially available and
distilled before use. Reactions were monitored by thin-
layer chromatography (TLC) on silica gel plates (60
F254), visualizing with ultraviolet. Infra red spectra (KBr)
were recorded on FT-IR 5300 spectrophotometer and
Perkin Elmer spectrum RXIFT-IR system (ν, cm1).
1HNMR spectra were recorded on Varian Gemini spec-
trophotometer (300 MHz) in DMSO-d6 or CDCl3 as sol-
vent. Proton chemical shifts (d) are relative to tetrame-
thylsilane (TMS, d = 0.00) as internal standard and ex-
pressed in ppm. Spin multiplicities are given as s
(singlet), d (doublet), t (triplet), and m (multiplet) as well
as b (broad). Coupling constants (J) are given in hertz.
Melting points were determined by using melting point
apparatus and are uncorrected. MS spectra were obtained
on a GC Ms-QP 1000 EX mass spectrometer at 70 eV.
Microanalyses were performed using a C H N S/O ana-
lyzer. Elemental data are within ±0.4% of the theoretical
values. All yields reported areunoptimized. The chemical
reagents used in synthesis were purchased from Fluka,
Sigma and Aldrich.
4.1.2. General Proce dure fo r Prepara ti on of
Compounds 2a,b
A warm solution of 5(6)-benzofuroxane 1a,b and the
appropriate acetoacetanilide (0.01 mol) in ethanol (50
mL) was stirred at room temperature in the presence of
catalytic amount of potassium carbonate. The yellow
products precipitated over a period of 2 - 4 h. The resid-
ual product was triturated with water, extracted with
ethylacetate then the organic solvent was dried over an-
hydrous sodium sulphate and removed in vacuo to give
yellow crystals that were recrystallized out from ethanol.
1) 2-N-(4-Fluorophenyl)-3-methyl-2-quinoxaline car-
boxamide 1,4-di-N-oxide 2a.
Yield (78%); mp 221˚C. IR (KBr, cm1): 1651 (C=O),
1575 (C=N), 3290 (NH), 1215, 1295 (NO). 1H NMR
spectrum (DMSO-d6), δ, ppm 2.63 (3H, s, CH3), 11.04
(1H, s, NH-D2O exchangable), 7.23 (2H, d, Ar-H, J = 8.7
Hz), 7.6 (2H, d, Ar-H, J = 8.7 Hz), 7.99 (2H, d, H5, H8),
8.52 (m, 2H, H6, H7). MS (m/z): 313 (M+), 281 (M+-O2),
202 (M+-4-F-Ph-NH), 171 (M+-O2, -4-F-Ph-NH), 129
(quinoxaline+). Found, %: C; H; N. C 61.47; H 3.66; N,
13.12. C16H12FN3O3. Calc., %: C, 61.34; H, 3.86; N, 13.41.
2) 7-chloro-2-N-(4-fluorophenyl)-3-methyl-2-quinox-
aline carboxamide 1,4-dioxide 2b.
Yield (85%); mp 243˚C. IR (KBr, cm1): 1670 (C=O),
1561 (C=N), 3211 (NH), 1334 (NO). MS (m/z): 349
(M+), 205 (M+-NH- C6H5-4-F, -O2). Found, %: C; H; N.
C 54.47; H 3.66; N, 13.01. C16H13ClFN3O3. Calc., %: C,
54.95; H, 3.75; N, 12.01.
4.1.3. General Proce dure fo r Prepara ti on of
Compounds 3a,b
A mixture of 5(6)-substituted benzofuroxane 1a,b and
cyanoacetamide (0.01 mol) was stirred for 10 min at 0˚C.
Over the cooled suspension, a solution of triethylamine
(5 drops) in dimethylformamide was added. The mixture
was allowed to stand at room temperature over 24 h and
then filtered off. The red solid product was filtered and
recrystallized from ethanol.
1) 3-Amino-2-quinoxalinecarboxamide 1,4-dioxide 3a.
Yield 71%; mp 260˚C - 265˚C. IR (KBr, cm1): 1660
(C=O), 1640 (C=N), 3388, 3224 (2NH2), 1254 (NO). 1H
NMR spectrum (DMSO-d6), δ, ppm 5.6 (s, 2H, NH2),
8.53 (s, 2H, CO-NH2), 7.26 - 8.2 (m, 4H, quinox-H).
Found, %: C; H; N. C 49.47; H 2.96; N, 25.40.
C9H8N4O3 C, 49.09; H, 3.66; N, 25.45.
2) 3-Amino-7-chloro-2-quinoxaline carboxamide 1,4-
dioxide 3b.
Yield 92%; mp. 270˚C - 271˚C. IR (KBr, cm1): 1715
(C=O), 1640 (C=N), 1319, 1340 (NO), 3356, 3454, 3512
(2NH2). 1H NMR spectrum (DMSO-d6), δ, ppm 5.76 (s,
2H, NH2), 8.60 (s, 2H, CO-NH2), 7.36-8.32 (m, 4H, qui-
nox-H). Found, %: C; H; N. C 42.47; H 2.66; N, 22.12.
C9H7ClN4O3 C, 42.45; H, 2.77; N, 22.00.
4.1.4. General Proce dure fo r Prepara ti on of
Compounds 4a-n
A warm solution of 5(6)-benzofuroxane 1a,b and the
appropriate cyanoacetanilide (0.01 mol) in ethanol (50
mL) was stirred at room temperature in the presence of
catalytic amount of potassium carbonate. The solutions
turned red and then they were stirred at room temperature
for 1 - 5 hr depending on the BFO and the cyanoacetani-
lides. The residue obtained was triturated with waterand
then extracted with dichloromethane. The organic phase
was dried with anhydrous sodium sulphate and filtered.
The solvent was removed in vacuo and the red-brown
precipitate was recrystallized out from ethanol.
1) 3-Amino-N-phenyl-2-quinoxaline carboxamide 1,4-
di-N-oxide 4a.
Yield (67%); mp 180˚C. IR (KBr, cm1): 1668 (C=O),
1563 (C=N), 3210 (NH), 3256, 3390 (NH2),1301 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 4.2 (s,2H,NH2),
7.1 (t, 1H, H4’), 7.26 (t, 2H, H3’ + H5’), 7.49 (d, 2H, H2
+ H6’), 7.85 - 7.97 (m, 2H, H6 + H7), 8.1 (d, 2H, H5 + H8),
10.79 (s, 1H, NH-D2O exchangeable) ppm. Found, %: C;
H; N. C 60.47; H 4.66; N, 18.12. C15H12N4O3. Calc., %:
C, 60.81; H, 4.08; N, 18.91.
2) 3-Amino-N-benzyl-2-quinoxaline carboxamide 1,4-
Open Access IJOC
D. H. SOLIMAN
70
di-N-oxide 4b.
Yield (62%); mp 192˚C. IR (KBr, cm1): 1659 (C=O),
1547 (C=N), 3230 - 33313 (NH, NH2), 1298 (NO). 1H
NMR spectrum (DMSO-d6), δ, ppm 4.32, 4.64 (2d, 2H,
N-CH2), 7.26 - 7.46 (m, 5H, phenyl-H), 7.52 - 7.55 (m,
2H, H6 + H7), 8.48 (s, 2H, NH2-D2O exchangeable), 8.61
(t, 1H, NH-D2O exchangeable) ppm. Found, %: C; H; N.
C 61.47; H 4.66; N, 18.12. C16H14N4O3. Calc., %: C,
61.93; H, 4.55; N, 18.06.
3) 3-Amino-N-(4-chlorophenyl)-2-quinoxaline carbox-
amide 1,4-di-N-oxide 4c.
Yield (83%); mp 209˚C. IR (KBr, cm1): 1673 (C=O),
1631 (C=N), 3269 (NH), 3347, 3401(NH2), 1245 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 7.05 (t, H6 + H7),
7.2 (d, 2H, H5 + H8), 7.74 (d, 2H, H3’ + H5’), 7.79 (d, 2H,
H2’ + H4’), 10.12 (s, 1H, NH-D2O exchangeable), 8.0 (s,
2H, NH2-D2O exchangeable) ppm. Found, %: C; H; N. C
54.37; H 3.66; N, 16.12. C15H11ClN4O3. Calc., %: C,
54.47; H, 3.35; N, 16.94.
4) 3-Amino-N-(4-fluorophen yl)-2-quinoxaline carbox-
amide 1,4-di-N-oxide 4d.
Yield (81%); mp 210˚C. IR (KBr, cm1): 1670 (C=O),
1621 (C=N), 3256 (NH), 3327, 3398 (NH2),1305 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 7.08 (t, 2H, H6 +
H7), 7.62 (d, 2H, H5 + H8), 7.74 (d, 2H, H3’ + H5’, 7.99
(d, 2H, H2’ + H4’), 10.10(s, 1H, NH-D2O exchangeable),
10.85 (br. s, 2H, NH2-D2O exchangeable) ppm. MS (m/z):
314 (M+), 282 (M+-O2), 172 (M+-O2-NH-C6H5-4-F,).
Found, %: C; H; N. C 57.47; H 3.66; N, 17.12.
C15H11FN4O3. Calc., %: C, 57.33; H, 3.53; N, 17.83.
5) 3-Amino-N-(4-methoxyphenyl)-2-quinoxaline car-
boxamide 1,4-di-N-oxide 4e.
Yield (76%); mp 195˚C. IR (KBr, cm1): 1662 (C=O),
1611 (C=N), 3250 - 3378 (NH, NH2), 1325 (NO). 1H
NMR spectrum (DMSO-d6), δ, ppm 3.77(s, 3H, OCH3),
3.89 (br. s, 2H, NH2-D2O exchangeable), 6.92 - 7.278 (m,
8H, Ar-H + quinox-H), 78.06(d, 2H, H2’ + H4’), 10.32 (s,
1H, NH-D2O exchangeable), ppm. Found, %: C; H; N. C
58.47; H 4.66; N, 17.12. C16H14N4O4. Calc., %: C, 58.89;
H, 4.32; N, 17.17.
6) 3-Amino-N-(4-ethoxyphenyl)-2-quinoxaline carbox-
amide 1,4-di-N-oxide 4f.
Yield (79%); mp 198˚C. IR (KBr, cm1): 1660 (C=O),
1598 (C=N), 3259 - 3421 (NH, NH2), 1355 (NO). 1H
NMR spectrum (DMSO-d6), δ, ppm 1.29 (t, 3H,
OCH2CH3), 4.02 (q, 2H, OCH2CH3), 6.97 (m, 2H, H6 +
H7), 7.28 (d, 2H, H3’ + H5’), 7.73 (d, 2H, H5 + H8), 8.04
(d, 2H, H2’ + H4’), 10.67(s, 1H, NH-D2O exchangeable),
12.12 (br. s, 2H, NH2-D2O exchangeable), ppm. Found,
%: C; H; N. C 59.47; H 4.66; N, 16.12. C17H16N4O4.
Calc., %: C, 59.99; H, 4.74; N, 16.46.
7) 3-Amino-N-(pyridine-4-yl)-2-quinoxaline carbox-
amide 1,4-di-N-oxide 4g.
Yield (59%); mp 211˚C - 214˚C. IR (KBr, cm1): 1663
(C=O), 1604 (C=N), 3278 - 3435 (NH, NH2), 1345 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 7.41 (d, 2H, H5 +
H8), 7.71(m, 2H, H6 + H7), 8.22(d, 2H, H3’ + H5’), 8.37
(d, 2H, H2’ + H4’), 10.18 (s, 1H, NH-D2O exchangeable),
12.46 (br. s, 2H, NH2-D2O exchangeable), ppm. Found,
%: C; H; N. C 56.47; H 3.66; N, 23.12. C14H11N5O3.
Calc., %: C, 56.56; H, 3.73; N, 23.56.
8) 3-Amino-7-Chloro -N-phenyl-2-quinoxaline carbox-
amide 1,4-di-N-oxide 4h.
Yield (83%); mp 223˚C - 224˚C. IR (KBr, cm1): 1670
(C=O), 1615 (C=N), 3221 (NH), 3266, 3421 (NH2), 1353
(NO). 1H NMR spectrum (DMSO-d6), δ, ppm 7.43 (m,
1H, H4’), 7.66 (m, 2H, H3’ + H5’), 7.89(d, 2H, H2’ + H6’),
7.92 (d, 1H, H5), 8.02 (s, 1H, H8), 8.29 (d, 1H, H6), 10.70
(s, 1H, NH-D2O exchangeable), 13.6 (s, 2H, NH2-D2O
exchangeable), ppm. Found, %: C; H; N. C 54.47; H 3.66;
N, 16.12. C15H11ClN4O3 C, 54.47; H, 3.35; N, 16.94.
9) 3-Amino-7-Chloro-N-benzyl-2-quinoxaline carbox-
amide 1,4-di-N-oxide 4i.
Yield (84%); mp 214˚C - 216˚C. IR (KBr, cm1): 1664
(C=O), 1572 (C=N), 3223 - 3345 (NH, NH2), 1331 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 4.63 (d, 2H,
N-CH2), 7.26 - 7.46, 7.79 (2m, 7H, phenyl-H, H5 + H6),
7.99 (s, 1H, H8), 8.48 (s, 2H, NH2-D2O exchangeable),
8.81 (t, 1H, NH-D2O exchangeable) ppm. Found, %: C,
55.34; H, 3.70; N, 16.12.C16H13ClN4O3. Calc., %: C,
55.74; H, 3.80; N, 16.25.
10) 3-Amino-7-Chloro-N-(4-chlorophenyl)-2-quinoxa-
line carboxamide 1,4-di-N-oxide 4j.
Yield (89%); mp 240˚C. IR (KBr, cm1): 1674 (C=O),
1587 (C=N), 3224 (NH), 3276, 3470 (NH2), 1335 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 7.46 (d, 1H, H2
+ H6’), 7.59(d, 1H, H6), 7.73 (d, 2H H3’ + H5’), 7.83 (s,
1H, H8), 7.88 (d, 1H, H5), 10.89 (s, 1H, NH-D2O
exchangeable), 13.69(s, 2H, NH2-D2O exchangeable),
ppm. MS (m/z): 364 (M+), 332(M+-O2), 191(M+-O2-NH-
C6H5-4-Cl-NH). Found, %: C 49.47; H 2.66; N, 15.12.
C15H10Cl2N4O3 C, 49.34; H, 2.76; N, 15.34.
11) 3-Amino-7-Chloro-N-(4-flourorophenyl)-2-quinoxa-
line carboxamide 1,4-di-N-oxide 4k.
Yield (91%); mp 244˚C. IR (KBr, cm1): 1669 (C=O),
1602 (C=N), 3210 - 3380 (NH, NH2), 1301 (NO). MS
(m/z): 348 (M+), 350 (M + 2), 332 (M+-O2), 221 (M+-O2-
NH-C6H5-4-F). Found, %: C; H; N. C 51.47; H 2.66; N,
16.12. C15H10ClFN4O3 C, 51.66; H, 2.89; N, 16.07.
12) 3-Amino-7-Chloro-N-(4-methoxyphenyl)-2-quinoxa-
linecarboxamide 1,4-di-N-oxide 4l.
Yield (82%); mp 219˚C. IR (KBr, cm1): 1664 (C=O),
1520 (C=N), 3298 (NH), 3620, 3589 (NH2), 1248 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 3.75 (s, 3H,
OCH3), 6.97 (d, 2H, H3’ + H5’), 7.62 (d, 2H, H2’ + H6’),
7.81 (s. 1H, H8), 8.00 (d, 1H, H5), 8.22 (D, 1H, H6),
10.27 (s, 1H, NH-D2O exchangeable), 13.60 (s, 2H, NH2-
D2O exchangeable), ppm. MS (m/z): 360 (M+), 362 (M +
Open Access IJOC
D. H. SOLIMAN 71
2), 206 (M+-O2-NH-C6H5-4-OCH3). Found, %: C; H; N.
C 53.47; H 3.66; N, 15.12. C16H13ClN4O4 C, 53.27; H,
3.63; N, 15.53.
13) 3-Amino-7-Chloro-N-(4-ethoxyphenyl)-2-quinoxa-
line carboxamide 1,4-di-N-oxide 4m.
Yield (84%); mp 219˚C - 220˚C. IR (KBr, cm1): 1663
(C=O), 1613 (C=N), 3255, 3389 (NH, NH2), 1351 (NO).
1H NMR spectrum (DMSO-d6), δ, ppm 3.75 (s, 3H,
OCH3), 6.97(d, 2H, H3’ + H5’), 7.62(d, 2H, H2’ + H6’),
7.81 (s. 1H, H8), 8.00 (d, 1H, H5), 8.22 (D, 1H, H6),
10.27 (s, 1H, NH-D2O exchangeable), 13.60 (s, 2H, NH2-
D2O exchangeable), ppm. MS (m/z): 360 (M+), 362 (M +
2), 206 (M+-O2-NH-C6H5-4-OCH3). Found, %: C; H; N.
C 53.07; H 3.26; N, 15.12. C16H13ClN4O4 C, 53.27; H,
3.63; N, 15.53.
14) 3-Amino-7-Chlo ro -N-(pyridin-4-yl)-2-quinoxaline
carboxamide 1,4-di-N-oxide 4n.
Yield (63%); mp 223˚C. IR (KBr, cm1): 1658 (C=O),
1554 (C=N), 3202 - 3325(NH, NH2), 1251 (NO). 1H
NMR spectrum (DMSO-d6), δ, ppm 7.03 (d, 1H, H5),
7.91 (d, 1H, H6), 8.14 (d, 2H, H2’ + H6’), 8.17 (s. 1H, H8),
8.34 (d, 2H, H3’ + H5’), 10.27 (s, 1H, NH-D2O ex-
changeable), 13.60 (s, 2H, NH2-D 2O exchangeable), ppm.
MS (m/z): 331 (M+), 333 (M + 2), 317 (M+-O), 302
(M+-O2), 208(M+-NH-pyridine), 192(M+-O2-NH-pyridine-
NH2). Found, %: C; H; N. C 49.77; H 2.96; N, 21.15.
C14H10ClN5O3C, 50.69; H, 3.04; N, 21.11.
4.1.5. 3,3-Dimethyl-1-Oxo-1,2,3,4-Tetrahydrophenazine
5,10-Dioxide 5
A mixture of 5(6)-substituted benzofuroxane 1a (0.01
mol) and dimidone (0.01 mol) in ethanol (50 mL) was
stirred and heated at 60˚C, while ammonia gas was bub-
bled into the solution for 1 hr. The solution was stirred at
room temperature for another 11 hr. The solvent was
then removed and finally a yellow precipitate was ob-
tained which was recrystallized out from ethanol.
Yield 75%; mp. 267˚C - 268˚C. IR (KBr, cm1): 1670
(C=O), 1627 (C=N), 1319, 1340 (NO). 1H NMR spec-
trum (DMSO-d6), δ, ppm 1.16, 1.22 (2s, 6H, 2CH3), 3.05
(s, 2H, H2), 3.34 (s, 2H, H4), 7.76 (m, 2H, H7), 7.86 (m,
2H, H8), 8.43 (d, 1H, H6), 8.61 (d, 1H, H9). Found, %: C;
H; N. C 65.17; H 5.36; N, 11.12. C14H14N2O3, C, 65.11;
H, 5.46; N, 10.85.
4.1.6. 11-Oxo-11H-inde no[1,2-b]Quinoxaline
5,10-Dioxide 6
A solution of 5(6)-substituted benzofuroxane 1a and in-
danedione (0.01 mol) inethanol (50 Ml) was stirred for
12 hr at room temperature in the presence of morpholine
as a catalyst. The red precipitate separated after standing
at room temperature for an additional 12 hr. The product
was finally filtered and recrystallized out from ethanol.
Yield 61%; mp. 222˚C - 225˚C. IR (KBr, cm1): 1715
(C=O), 1642 (C=N), 1321, 1345 (NO).1H NMR spectrum
(DMSO-d6), δ, ppm 7.68 (m, 2H, H7 + H8), 7.73 (d, 2H,
H6 + H9), 8.00 (m, 1H, H3), 8.03 (m, 1H, H2), 8.35 (d, 2H,
H4), 8.85 (d, 1H, H1), MS (m/z): 264 (M+), 248 (M+-O),
232 (M+-O2), 204(M+-CO). Found, %: C; H; N. C 69.01;
H 2.66; N, 11.12. C15H8N2O3C, 68.18; H, 3.05; N, 10.60.
4.2. Antimicrobial and Antifungal Assays
Antimicrobial activity was determined using the agar
well diffusion assay method as described by [20]. The
tested organisms were subcultured on nutrient agar me-
dium (Oxoid laboratories, UK) for bacteria and Saboroud
dextrose agar (Oxoid laboratories, UK) for fungi. Am-
picillin and Gentamycin were used as a positive control
for bacterial strains. Amphotericin B was used as a posi-
tive control for fungi. The plates were done in triplicate.
Bacterial cultures were incubated at 37˚C for 24 h while
the other fungal cultures were incubated at (25˚C - 30˚C)
for 3 - 7 days. Antimicrobial activity was determined by
measurement zone of inhibition [21].
Determination of MIC
The minimum inhibitory concentration (MIC) of the
samples was estimated for each of the tested organisms
in triplicates. Varying concentrations of the samples
(1000 - 0.007 µg/ml), nutrient broth were added and then
a loopful of the test organism previously diluted to 0.5
McFarland turbidity standard was introduced to the tubes.
A tube containing broth media only was seeded with the
test organisms to serve as control. Tubes containing
tested organisms cultures were then incubated at 37˚C for
24 h while the other fungal cultures were incubated at
(25˚C - 30˚C) for 3 - 7 days. The tubes were then exam-
ined for growth by observing for turbidity [22].
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